6&#34;-amino-6&#34;-deoxygalactosylceramides

ABSTRACT

This invention relates to galactosylceramide compounds.

This application is a Continuation application of U.S. Ser. No.13/196,631, filed 2 Aug. 2011, which is a Continuation application ofU.S. Ser. No. 12/625,048, filed 24 Nov. 2009, which issued as U.S. Pat.No. 7,989,423 on 2 Aug. 2011, which is a Divisional application of U.S.Ser. No. 10/550,165, filed 21 Jul. 2006, which issued as U.S. Pat. No.7,645,873 on 12 Jan. 2010, which is a National Stage Application ofPCT/US2003/008530, filed 20 Mar. 2003, and which applications areincorporated herein by reference. To the extent appropriate, a claim ofpriority is made to each of the above disclosed applications.

TECHNICAL FIELD

This invention relates to galactosylceramide compounds.

BACKGROUND

Peptide antigen presentation via major histacompatability complexes haslong been recognized as a central element in adaptive immune responses.Recently, a parallel pathway that can elicit potent immune responses hasbegun to be elucidated. This pathway involves the presentation ofglycolipids by CD1 proteins and is believed to be responsible for aportion of the innate immunity of mammals to bacteria.

The CD1 locus of mammals includes five distinct isotypes, CD1a, CD1b,CD1c, CD1d, and CD1e. These nonpolymorphic, membrane-bound proteins arecharacterized by their ability to present classes of glycolipids to Tcells. The CD1d member of the gene family has been characterized by itsability to bind and present α-galactosylceramides to natural killer Tcells (NKT cells).

Complex formation between glycolipid-loaded CD1d proteins and T cellreceptors can subsequently lead to the stimulation of T cells. T cellstimulation initiates, inter alia, the cellular production of certainkey immunoresponsive biochemicals.

SUMMARY

This invention relates to 6″-amino-6″-deoxygalactosylceramide compoundsand their methods of use.

One aspect of this invention features compounds of Formula (I):

wherein,

R₁ is:

-   -   (i) hydrogen; or    -   (ii) —SO₂R₁₀,    -   wherein R₁₀ is:    -   halo; hydroxy; OR₁₁; OR₁₂; amino; NHR₁₁; N(R₁₁)₂; NHR₁₂;        N(R₁₂)₂; aralkylamino; or    -   C₁-C₁₂ alkyl optionally substituted with halo, hydroxy, oxo,        nitro, OR₁₁, OR₁₂, acyloxy, amino, NHR₁₁, N(R₁₁)₂, NHR₁₂,        N(R₁₂)₂, aralkylamino, mercapto, thioalkoxy, S(O)R₁₁, S(O)R₁₂,        SO₂R₁₁, SO₂R₁₂, NHSO₂R₁₁, NHSO₂R₁₂, sulfate, phosphate, cyano,        carboxyl, C(O)R₁₁, C(O)R₁₂, C(O)OR₁₁, C(O)NH₂, C(O)NHR₁₁,        C(O)N(R₁₁)₂, C₃-C₁₀ cycloalkyl containing 0-3 R₁₃, C₃-C₁₀        heterocyclyl containing 0-3 R₁₃, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        C₅-C₁₀ cycloalkenyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₂₀ aryl        containing 0-3 R₁₄, or heteroaryl containing 0-3 R₁₄; or    -   C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocyclyl, C₅-C₁₀ cycloalkenyl, or        C₅-C₁₀ heterocycloalkenyl optionally substituted with one or        more halo, hydroxy, oxo, OR₁₁, OR₁₂, acyloxy, nitro, amino,        NHR₁₁, N(R₁₁)₂, NHR₁₂, N(R₁₂)₂, aralkylamino, mercapto,        thioalkoxy, S(O)R₁₁, S(O)R₁₂, SO₂R₁₁, SO₂R₁₂, NHSO₂R₁₁,        NHSO₂R₁₂, sulfate, phosphate, cyano, carboxyl, C(O)R₁₁, C(O)R₁₂,        C(O)OR₁₁, C(O)NH₂, C(O)NHR₁₁, C(O)N(R₁₁)₂, alkyl, haloalkyl,        C₃-C₁₀ cycloalkyl containing 0-3 R₁₃, C₃-C₁₀ heterocyclyl        containing 0-3 R₁₃, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₅-C₁₀        cycloalkenyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₂₀ aryl heteroaryl        containing 0-3 R₁₄, or C₆-C₂₀ heteroaryl containing 0-3 R₁₄; or    -   C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, or heteroaryl optionally        substituted with one or more halo, hydroxy, OR₁₁, OR₁₂, acyloxy,        nitro, amino, NHR₁₁, N(R₁₁)₂, NHR₁₂, N(R₁₂)₂, aralkylamino,        mercapto, thioalkoxy, S(O)R₁₁, S(O)R₁₂, SO₂R₁₁, SO₂R₁₂,        NHSO₂R₁₁, NHSO₂R₁₂, sulfate, phosphate, cyano, carboxyl,        C(O)R₁₁, C(O)R₁₂, C(O)OR₁₁, C(O)NH₂, C(O)NHR₁₁, C(O)N(R₁₁)₂,        alkyl, haloalkyl, C₃-C₁₀ cycloalkyl containing 0-3 R₁₃, C₃-C₁₀        heterocyclyl containing 0-3 R₁₃, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        C₅-C₁₀ cycloalkenyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₂₀ aryl        containing 0-3 R₁₄, or C₆-C₂₀ heteroaryl containing 0-3 R₁₄; or    -   (iii) —C(O)R₁₀, wherein R₁₀ is defined as above; or    -   (iv) —C(R₁₀)₂(R₁₅), wherein R₁₀ is defined as above; R₁₅ is        hydrogen, R₁₀, or R₁₅ and R₂ taken together forms a double bond        between the carbon and nitrogen atoms to which they are        attached; or    -   (v) R₁ and R₂ taken together forms a heterocyclyl of 3-10 ring        atoms optionally substituted with R₁₀;

R₂ is hydrogen, or R₂ and R₁₅ taken together forms a double bond betweenthe carbon and nitrogen atoms to which they are attached, or R₂ and R₁taken together forms a heterocyclyl of 3-10 ring atoms optionallysubstituted with R₁₀;

R₃, R₄, R₅, R₆, and R₇ are each independently hydrogen, C₁-C₆ alkyl,C₆-C₁₂ aralkyl, or C₁-C₆ acyl;

R₈ is —(CH₂)_(x)CH₃;

R₉ is a linear or branched C₃-C₁₀₀ alkyl;

R₁₁ is C₁-C₂₀ alkyl optionally substituted with halo, hydroxy, alkoxy,amino, alkylamino, dialkylamino, sulfate, or phosphate;

R₁₂ is aryl optionally substituted with halo, haloalkyl, hydroxy,alkoxy, nitro, amino, alkylamino, dialkylamino, sulfate, or phosphate;

Each R₁₃ is independently halo, haloalkyl, hydroxy, alkoxy, oxo, amino,alkylamino, dialkylamino, sulfate, or phosphate;

Each R₁₄ is independently halo, haloalkyl, hydroxy, alkoxy, nitro,amino, alkylamino, dialkylamino, sulfate, or phosphate; and

x is 1-100.

Referring to Formula (I) above, a subset of compounds described aboveare those in which x is 24 and R₉ is n-tetradecyl.

In some embodiments, R₁ is SO₂R₁₀ and R₁₀ can be aryl substituted withN(R₁₁)₂, e.g.:

In other embodiments, R₁ is C(O)R₁₀ and R₁₀ can be C₁-C₆ alkylsubstituted with halo, hydroxy, oxo, nitro, OR₁₁, OR₁₂, acyloxy, amino,NHR₁₁, N(R₁₁)₂, NHR₁₂, N(R₁₂)₂, aralkylamino, mercapto, thioalkoxy,S(O)R₁₁, S(O)R₁₂, SO₂R₁₁, SO₂R₁₂, NHSO₂R₁₁, NHSO₂R₁₂, sulfate,phosphate, cyano, carboxyl, C(O)R₁₁, C(O)R₁₂, C(O)OR₁₁, C(O)NH₂,C(O)NHR₁₁, C(O)N(R₁₁)₂, C₃-C₁₀ cycloalkyl containing 0-3 R₁₃, C₃-C₁₀heterocyclyl containing 0-3 R₁₃, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₅-C₁₀cycloalkenyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₂₀ aryl containing 0-3 R₁₄,or C₆-C₂₀ heteroaryl containing 0-3 R₁₄.

In certain embodiments, R₁₀ can be C₁-C₆ alkyl substituted withNHSO₂R₁₂, in which R₁₂ is e.g.:

In certain embodiments, R₁₀ can be alkyl substituted with C(O)R₁₂, inwhich e.g. R₁₂ is:

In certain embodiments, R₁₀ can be alkyl substituted with C₅-C₁₀heterocyclyl containing 0-3 R₁₃ in which the heterocyclyl is e.g.:

In another aspect, this invention relates to a probe for observingglycolipid association with CD1d and NKT cell receptors during NKT cellstimulation having a compound of Formula (II):

wherein:

-   -   X is —SO₂—, —C(O)—, or absent;    -   Y is a linker group; and    -   Z is a reporter group.

In a further aspect, this invention relates to a method of quantifyingglycolipid association with CD1d and NKT cell receptors during NKT cellstimulation including: (i) contacting a compound of Formula (II) with aCD1d protein; (ii) allowing the compound to associate with the CD1dprotein; (iii) measuring fluorescence emitted by the compound duringsteps (i) and (ii) to provide one or more pre-NKT cell contactfluorescence measurements; (iv) contacting the compound and CD1d proteinwith an NKT cell line; (v) measuring fluorescence emitted by thecompound during step (iv) to provide one or more NKT cell contactfluorescence measurements.

Embodiments can include one or more of the following features.

Step (v) can be repeated over time.

The method can further include the step of comparing the fluorescencemeasurements in steps (iii) and (v).

In one aspect, this invention features a method of stimulating NKTcells, which includes contacting an NKT cell with a compound of Formula(I) and a CD1 protein.

The protein can be a CD1d protein.

In another aspect, this invention features a method of stimulating theimmune system of a subject (e.g., mammal, human, dog, horse, cat) inneed of such stimulation, the method includes administering a compoundof Formula (I) to the subject.

In a further aspect, this invention features a method of treating anautoimmune disease in a subject (e.g., mammal, human, dog, horse, cat)in need of such treatment, the method includes administering aneffective amount of a compound of Formula (I).

The subject can be a mammal, preferably a human. Identifying a subjectin need of such treatment can be in the judgement of a subject or ahealth care professional and can be subjective (e.g., opinion) orobjective (e.g., measurable by a test or diagnostic method).

In one aspect, this invention relates to a method of making a compounddescribed herein. In some embodiments, the method can be a method ofmaking a compound of Formula (I) including: (i) converting a compound ofFormula (III) to a compound of Formula (IV):

and (ii) contacting a compound of Formula (IV) with R₁-LG to afford acompound of Formula (I), wherein:

R₁ is:

-   -   (i) —SO₂R₁₀,    -   wherein R₁₀ is:    -   halo; hydroxy; OR₁₁; OR₁₂; amino; NHR₁₁; N(R₁₁)₂; NHR₁₂;        N(R₁₂)₂; aralkylamino; or    -   C₁-C₁₂ alkyl optionally substituted with halo, hydroxy, oxo,        nitro, OR₁₁, OR₁₂, acyloxy, amino, NHR₁₁, N(R₁₁)₂, NHR₁₂,        N(R₁₂)₂, aralkylamino, mercapto, thioalkoxy, S(O)R₁₁, S(O)R₁₂,        SO₂R₁₁, SO₂R₁₂, NHSO₂R₁₁, NHSO₂R₁₂, sulfate, phosphate, cyano,        carboxyl, C(O)R₁₁, C(O)R₁₂, C(O)OR₁₁, C(O)NH₂, C(O)NHR₁₁,        C(O)N(R₁₁)₂, C₃-C₁₀ cycloalkyl containing 0-3 R₁₃, C₃-C₁₀        heterocyclyl containing 0-3 R₁₃, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        C₅-C₁₀ cycloalkenyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₂₀ aryl        containing 0-3 R₁₄, or C₆-C₂₀ heteroaryl containing 0-3 R₁₄; or    -   C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocyclyl, C₅-C₁₀ cycloalkenyl, or        C₅-C₁₀ heterocycloalkenyl optionally substituted with one or        more halo, hydroxy, oxo, OR₁₁, OR₁₂, acyloxy, nitro, amino,        NHR₁₁, N(R₁₁)₂, NHR₁₂, N(R₁₂)₂, aralkylamino, mercapto,        thioalkoxy, S(O)R₁₁, S(O)R₁₂, SO₂R₁₁, SO₂R₁₂, NHSO₂R₁₁,        NHSO₂R₁₂, sulfate, phosphate, cyano, carboxyl, C(O)R₁₁, C(O)R₁₂,        C(O)OR₁₁, C(O)NH₂, C(O)NHR₁₁, C(O)N(R₁₁)₂, alkyl, haloalkyl,        C₃-C₁₀ cycloalkyl containing 0-3 R₁₃, C₃-C₁₀ heterocyclyl        containing 0-3 R₁₃, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₅-C₁₀        cycloalkenyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₂₀ aryl containing        0-3 R₁₄, or C₆-C₂₀ heteroaryl containing 0-3 R₁₄; or    -   C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, or heteroaryl optionally        substituted with one or more halo, hydroxy, OR₁₁, OR₁₂, acyloxy,        nitro, amino, NHR₁₁, N(R₁₁)₂, NHR₁₂, N(R₁₂)₂, aralkylamino,        mercapto, thioalkoxy, S(O)R₁₁, S(O)R₁₂, SO₂R₁₁, SO₂R₁₂,        NHSO₂R₁₁, NHSO₂R₁₂, sulfate, phosphate, cyano, carboxyl,        C(O)R₁₁, C(O)R₁₂, C(O)OR₁₁, C(O)NH₂, C(O)NHR₁₁, C(O)N(R₁₁)₂,        alkyl, haloalkyl, C₃-C₁₀ cycloalkyl containing 0-3 R₁₃, C₃-C₁₀        heterocyclyl containing 0-3 R₁₃, C₂-C₆ alkenyl, C₂-C₆ alkynyl,        C₅-C₁₀ cycloalkenyl, C₅-C₁₀ heterocycloalkenyl, C₆-C₂₀ aryl        containing 0-3 R₁₄, or C₆-C₂₀ heteroaryl containing 0-3 R₁₄; or    -   (ii) —C(O)R₁₀, wherein R₁₀ is defined as above; or    -   (iii) —C(R₁₀)₂(R₁₅), wherein R₁₀ is defined as above; R₁₅ is        hydrogen, R₁₀, or R₁₅ and R₂ taken together forms a double bond        between the carbon and nitrogen atoms to which they are        attached; or

R₃, R₄, R₅, R₆, and R₇ are each independently hydrogen, C₁-C₆ alkyl,C₆-C₁₂ aralkyl, or C₁-C₆ acyl;

R₈ is —(CH₂)_(x)CH₃;

R₉ is a linear or branched C₃-C₁₀₀ alkyl;

R₁₁ is C₁-C₂₀ alkyl optionally substituted with halo, hydroxy, alkoxy,amino, alkylamino, dialkylamino, sulfate, or phosphate;

R₁₂ is aryl optionally substituted with halo, haloalkyl, hydroxy,alkoxy, nitro, amino, alkylamino, dialkylamino, sulfate, or phosphate;

Each R₁₃ is independently halo, haloalkyl, hydroxy, alkoxy, oxo, amino,alkylamino, dialkylamino, sulfate, or phosphate;

Each R₁₄ is independently halo, haloalkyl, hydroxy, alkoxy, nitro,amino, alkylamino, dialkylamino, sulfate, or phosphate;

x is 1-100;

LG is halo, —OSO₂R₁₆, B(OH)₂, or

and

R₁₆ is alkyl, haloalkyl or aryl optionally substituted with alkyl, haloor nitro.

In another aspect, this invention features a pharmaceutical compositionincluding a compound of Formula (I) and a pharmaceutically acceptablecarrier.

Also within the scope of this invention is a composition containing oneor more of the compounds described herein for use in treating cancer orautoimmune disorders, diseases, or disease symptoms, including any ofthose delineated herein, and the use of such a composition for themanufacture of a medicament for the just-mentioned use.

Also within the scope of this invention is a packaged product. Thepackaged product includes a container, one of the aforementionedcompounds in the container, and a legend (e.g., a label or insert)associated with the container and indicating administration of thecompound for treating cancer or autoimmune disorders, diseases, ordisease symptoms, including any of those delineated herein.

Embodiments of the invention may have one or more of the followingadvantages. For example, the replacement of the parent sugar'sC₆″-hydroxyl by the more reactive amino group allows for the efficientsynthesis of a more expansive range ofC₆″-amino-C₆″-deoxygalactosylceramides. Further, having thederivitization handle situated at C₆″ allows α-galactosylceramides to bemodified without significantly altering their binding to the proteinsand receptors involved in T cell stimulation because the C₆″-aminosubstituents are sufficiently distanced from the lipid portion of themolecule, which is known to engage in relatively strong interactionswith the deep hydrophobic pockets of the CD1d proteins.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows NKT cell stimulatory activity of fluorophore-appendedC₆″-amino-C₆″-deoxygalactosylceramides 8, 10, 12.

FIG. 2 shows NKT cell stimulatory activity of biotin-appendedC₆″-amino-C₆″-deoxygalactosylceramide 13.

DETAILED DESCRIPTION

As used herein, the term “halo” or “halogen” refers to any radical offluorine, chlorine, bromine or iodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁˜C₁₂ alkyl indicates that the group may have from1 to 12 (inclusive) carbon atoms in it. The terms “arylalkyl” or“aralkyl” refer to an alkyl moiety in which an alkyl hydrogen atom isreplaced by an aryl group. Examples of “arylalkyl” or “aralkyl” includebenzyl and 9-fluorenyl groups.

The terms “alkylamino” and “dialkylamino” refer to —NH(alkyl) and—NH(alkyl)₂ radicals respectively. The term “aralkylamino” refers to a—NH(aralkyl) radical. The term “alkoxy” refers to an —O-alkyl radical.The term “mercapto” refers to an SH radical. The term “thioalkoxy”refers to an —S-alkyl radical.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom capable of substitutioncan be substituted by a substituent. Examples of aryl moieties include,but are not limited to, phenyl, naphthyl, and anthracenyl.

The term “cycloalkyl” as employed herein includes saturated cyclic,bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12carbons, wherein any ring atom capable of substitution can besubstituted by a substituent. Examples of cycloalkyl moieties include,but are not limited to, cyclohexyl and adamantyl.

The term “heterocyclyl” refers to a nonaromatic 3-10 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively), whereinany ring atom capable of substitution can be substituted by asubstituent.

The term “cycloalkenyl” as employed herein includes partiallyunsaturated, nonaromatic, cyclic, bicyclic, tricyclic, or polycyclichydrocarbon groups having 5 to 12 carbons, preferably 5 to 8 carbons,wherein any ring atom capable of substitution can be substituted by asubstituent. Examples of cycloalkyl moieties include, but are notlimited to cyclohexenyl, cyclohexadienyl, or norbornenyl.

The term “heterocycloalkenyl” refers to a partially saturated,nonaromatic 5-10 membered monocyclic, 8-12 membered bicyclic, or 11-14membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, saidheteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6,or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,respectively), wherein any ring atom capable of substitution can besubstituted by a substituent.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein any ring atomcapable of substitution can be substituted by a substituent.

The term “oxo” refers to an oxygen atom, which forms a carbonyl whenattached to carbon, an N-oxide when attached to nitrogen, and asulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl,arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent,any of which may be further substituted by substituents.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl,cycloalkenyl, aryl, or heteroaryl group at any atom of that group.Suitable substituents include, without limitation, alkyl, alkenyl,alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H, sulfate,phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy,ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl),S(O)_(n)alkyl (where n is 0-2), S(O)_(n) aryl (where n is 0-2), S(O)_(n)heteroaryl (where n is 0-2), S(O)_(n) heterocyclyl (where n is 0-2),amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, andcombinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide(mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof),sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinationsthereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstitutedheterocyclyl, and unsubstituted cycloalkyl. In one aspect, thesubstituents on a group are independently any one single, or any subsetof the aforementioned substituents.

The term “treating” or “treated” refers to administering a compounddescribed herein to a subject with the purpose to cure, heal, alleviate,relieve, alter, remedy, ameliorate, improve, or affect a disease, thesymptoms of the disease or the predisposition toward the disease.

“An effective amount” refers to an amount of a compound that confers atherapeutic effect on the treated subject. The therapeutic effect may beobjective (i.e., measurable by some test or marker) or subjective (i.e.,subject gives an indication of or feels an effect). An effective amountof the compound described above may range from about 0.1 mg/Kg to about500 mg/Kg, alternatively from about 1 to about 50 mg/Kg. Effective doseswill also vary depending on route of administration, as well as thepossibility of co-usage with other agents.

The term “mammal” includes organisms, which include mice, rats, cows,sheep, pigs, goats, and horses, monkeys, dogs, cats, and preferablyhumans.

Structure of 6″-amino-6″-deoxygalactosylceramide compounds

In general, the 6″-amino-6″-deoxygalactosylceramide compounds include a“glyco” portion and a “lipid” portion as indicated in Formula (V). Thetwo portions are appended to one another via an α-glycosidic bondbetween the anomeric carbon, C_(a), of the “glyco” portion and theoxygen (bolded) bound to C₁ of the “lipid” portion.

The lipid portion is a chain of carbon atoms having functionalized andunfuctionalized segments. The functionalized segment includes carbonsC₁-C₄. The functionalized segment terminus C₁ is the carbon throughwhich the lipid portion is appended to the glyco portion of themolecule. The remaining members of this segment, C₂, C₃, and C₄, eachcontain a heteroatom substituent. The C₂ nitrogen is acylated withC(O)R₈, in which R₈ is a hydrocarbon chain represented by the formula(CH₂)_(x)CH₃. The hydrocarbon chain R₈ can have 1 to 100 methylene (CH₂)groups (e.g., 1 to 75 CH₂ groups, 1 to 50 CH₂ groups, 1 to 25 CH₂groups, 1 to 20 CH₂ groups, 1 to 15 CH₂ groups, 1 to 10 CH₂ groups, or 1to 5 CH₂ groups). In certain embodiments, R₈ contains 24 CH₂ groups. Theoxygens on C₃, and C₄, may be substituted with hydrogen, alkyl, aralkyl,trisubstituted silyl, or acyl groups. In some embodiments, R₅ and R₆ arehydrogen, and in other embodiments, they are tert-butyldimethylsilyl(TBS).

The unfunctionalized segment is represented by R₉, which can include anybranched or unbranched alkyl group containing 3-100 carbons atoms (e.g,3-75 carbons atoms, 3-50 carbons atoms, 3-25 carbons atoms, 3-20 carbonsatoms, 3-15 carbons atoms, or 3-10 carbons atoms). In certainembodiments, R₉ contains an unbranched alkyl group composed of 14 carbonatoms.

The glyco portion is a derivative of α-D-galactose. Each of R₃, R₄, andR₅ may be hydrogen, alkyl, aralkyl or acyl groups. When R₃-R₅ aresubstituted with a group other than hydrogen, the group is preferablyone that is readily removed using carbohydrate deprotection chemistriesthat are well known in the art. In certain embodiments, these groupsinclude methyl, benzyl or acetyl.

The C₆″-hydroxyl group of the parent sugar is replaced by a substituted(R₁ and/or R₂ are substituents other than hydrogen) or unsubstituted (R₁and R₂ are both hydrogen) amino group.

In certain embodiments, the C₆″-amino group is monosubstituted(R₁=substitutent and R₂=H). For example, the C₆″-nitrogen can form partof a sulfonamide (R₁=—SO₂R) or amide (R₁=C(O)R group. R may includee.g., substituted or unsubstituted alkyl, cycloalkyl, aryl, heteroaryl,etc. These groups can be formed upon the reaction of the unsubstitutedamino group with e.g., the corresponding sulfonyl halide or activatedacyl derivative. Alternatively, the nitrogen may form part of asecondary alkyl-alkyl or alkyl-aryl amino group (e.g., R₁=substituted orunsubstituted alkyl or aryl). Introduction of an alkyl group at R₁ canbe carried out e.g., by first exposing the unsubstituted amino group toa carbonyl compound and then performing a reductive alkylation on theresulting, intermediary aldimine or ketenimine. Introduction of an arylgroup at R₁ can be accomplished e.g., by transition metal mediatedcoupling between the unsubstituted amino group and an aryl halide ortriflate.

In other embodiments, the C₆″-amino group is disubstituted (R₁ and R₂substituent). In some embodiments, R₁ and R₂ may form a cyclic structurein which one or more of the ring atoms may be a heteroatom (e.g., N, O,or S). Further, any one of the ring atoms may be substituted with e.g.,halo, hydroxy, alkyl, haloalkyl, aryl, herteroaryl, etc.

In some embodiments, the C₆″-nitrogen can form part of an imino group,i.e., C═N. The imino group can be an aldimine, which may be obtained ina condensation reaction between the C₆″-amino group and a substituted orunsubstituted alkyl or aryl aldehyde. Similarly, the imino group can bea ketenimine, which may be obtained in a condensation reaction betweenthe C₆-amino group and a substituted or unsubstituted dialkyl ketone, adiarylketone, aryl-alkyl ketone, etc.

In certain embodiments, it can be advantageous for a reporter group tobe linked either directly or indirectly to the C6″-amino group. Whilenot wishing to be bound by theory, it believed that labellinggalactosylceramides with fluorophores or other small molecules (e.g.,biotin) would allow observation of the compounds at low concentrationsand/or provide a means of quantifying association with CD1d and NKT cellreceptors. Thus compounds containing reporter groups could be useful asprobes for determining e.g., specific structural requirements forglycolipid binding by CD1d and T cell receptors.

In some embodiments, probes are compounds in which the reporter groupmay be directly attached to the C₆″-amino group in either a covalent ornoncovalent manner. In other embodiments, the reporter group may beindirectly attached to the C₆-amino group via covalent or noncovalentlinkages. For example, when the reporter group is indirectly attached,the C₆″-amino group can be attached to a moiety —X—Y—Z, wherein X is—SO2-, —C(O)—, or absent; Y is a linker group, and Z is a reportergroup.

The linker group Y can be any carbon-containing chain or ring. Forexample, the linker can be —(CH₂)_(t)—, in which the chain optionallycontains one or more terminal heteroatoms (e.g., N, O, S), and/or one ormore heteroatoms, rings, double bonds, triple bonds that are insertedinto the chain. The value of “t” can be 1-100. The linker may also beone ring, or a series of two or more rings.

The reporter groups may be selected as desired. Selection of thereporter groups is within skill of the art. Examples of reporter groupsinclude labelling reagents, e.g., radiolabelled moieties, functionalsmall molecules, e.g., biotin, or fluorophores e.g., acridines, Cy5.5™,Dabcyl, Dansyl, Fluorescien, Oregon Green 488, Prodan, Tamra, etc.Representative reporter groups may be selected and obtained from e.g.,Molecular Probes, Inc. (www.molecularprobes.com).

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., therapeutic or prophylacticadministration to a subject).

Synthesis of 6″-amino-6″-deoxygalactosylceramide compounds

The synthesis of 6″-amino-6″-deoxygalactosylceramides may be carried outusing conventional methods including those described herein forexemplary compound 1 (R₃-R₇=H; R₈=(CH₂)₂₄CH₃; and R₉=C₁₄H₂₉). Ingeneral, compound 1 may be obtained from the reaction between compound 2and a desired electrophile, e.g., R₁-leaving group, as shown below.

The synthesis of compound 2 can be carried out as follows. The aminefunctionality was incorporated early onto the carbohydrate as the azideusing the procedure of Corey et al., J. Am. Chem. Soc. 1984, 106, 3682.(3, Scheme 1). Following the procedure of Singh, P. P., et al.,Carbohyd. Res. 1970, 12, 261, the acetonides were hydrolyzed withconcomitant methylgalactoside formation and benzyl ethers at C2, C3 andC4 were formed giving 4. The methoxy group was then replaced by anacetoxy group, followed by conversion to the anomeric fluoride (5)according to the methods of Davis, N. J., et al., J. Chem. Soc. PerkinTrans. 1 1994, 359 and Hayashi, M., et al., Chem. Lett. 1984, 1747respectively. Glycosyl bond formation with compound 6 gave 7 viaHashimoto, et al., Tetrahedron Lett. 1984, 25, 1379. Compound 6,(2S,3S,4R)-3,4-bis-t-butyldimethylsisyloxy-2-hexaosanoylamino-4-octadecanol,was prepared by coupling of phytosphingosine (Avanti Polar Lipids) withhexacosanoic acid using 2-dimethylaminoisopropyl chloride hydrochloride(DIC) and 1-hydroxybenzotriazole (HOBT), followed by theprotection/deprotection scheme reported by Takikawa, et al., Tetrahedron1998, 54, 3141. The silyl protecting groups were removed (Takikawa, etal.) followed by reduction of the azide (Vaultier, M. et al.,Tetrahedron Lett. 1983, 24, 763) and removal of the benzyl groups giving2 (Sakai, T. et al., J. Med. Chem. 1998, 41, 650).

-   -   Reagents (yields in parentheses): a) AcCl, MeOH (86% yield). b)        BnBr, 18-crown-6, NaH, THF (95% yield). c) AcOH, H₂SO₄ (84%        yield). d) HF.pyridine, pyridine (78% yield). e) MS 4 Å, AgClO₄,        SnCl₂, THF (44% yield). f) TBAF, THF (81% yield). g) PPh₃/H₂O,        THF (quant. yield). h) NH₃/Na, −78° C. (53% yield).

Reaction of 2 with acid chlorides and N-hydroxysuccinimidyl (NHS) esterscan provide reasonable yields of the corresponding amides. For example,compounds 8 and 10 were prepared from 2 and dansyl chloride and 9respectively (Scheme 2). The latter compound is a dansyl amide tetheredto an N-hydroxysuccinimidyl ester (Wang, F.; Schwabacher, A. W. J. Org.Chem. 1999, 64, 8922). NHS ester 11 can be prepared from4-(6-methoxy-[2]naphthyl)-4-oxo-butyric acid (Khan, M. et al., Indian J.Chem. Sect. B 2000, 39, 614) by nucleophilic displacement of the methoxygroup with lithium dimethylamide (Weber, G., et al., Biochemistry 1979,18, 3075) followed by reaction with dicyclohexylcarbodiimide (DCC) andN-hydroxysuccinimide. Reaction of NHS ester 11 with 2 gave 12 in 46%yield. Similarly, reaction of 2 with the N-hydroxysuccinimidyl ester ofbiotin gave 13 in 52% yield (Scheme 3). Glycosylceramides can exhibitrelatively limited solubility in many organic solvents, and thisrelatively high insolubility may result in loss of yield, e.g., duringpurification.

The synthesized 6″-amino-6″-deoxygalactosylceramide compounds can beseparated from a reaction mixture and further purified by a method suchas column chromatography, high pressure liquid chromatography, orrecrystallization. As can be appreciated by the skilled artisan, furthermethods of synthesizing the compounds of the formulae herein will beevident to those of ordinary skill in the art. Additionally, the varioussynthetic steps may be performed in an alternate sequence or order togive the desired compounds. Synthetic chemistry transformations andprotecting group methodologies (protection and deprotection) useful insynthesizing the compounds described herein are known in the art andinclude, for example, those such as described in R. Larock,Comprehensive Organic Transformations, VCH Publishers (1989); T. W.Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d.Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser andFieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); andL. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995), and subsequent editions thereof.

The 6″-amino-6″-deoxygalactosylceramide compounds of this invention maycontain one or more asymmetric centers and thus occur as racemates andracemic mixtures, single enantiomers, individual diastereomers anddiastereomeric mixtures. All such isomeric forms of these compounds areexpressly included in the present invention. The compounds of thisinvention may also be represented in multiple tautomeric forms, in suchinstances, the invention expressly includes all tautomeric forms of thecompounds described herein (e.g., alkylation of a ring system may resultin alkylation at multiple sites, the invention expressly includes allsuch reaction products). All such isomeric forms of such compounds areexpressly included in the present invention. All crystal forms of thecompounds described herein are expressly included in the presentinvention.

The 6″-amino-6″-deoxygalactosylceramide compounds of this inventioninclude the compounds themselves, as well as their salts and theirprodrugs, if applicable. A salt, for example, can be formed between ananion and a positively charged substituent (e.g., amino) on a6″-amino-6″-deoxygalactosylceramide compound. Suitable anions includechloride, bromide, iodide, sulfate, nitrate, phosphate, citrate,methanesulfonate, trifluoroacetate, and acetate. Likewise, a salt canalso be formed between a cation and a negatively charged substituent(e.g., carboxylate) on a 6″-amino-6″-deoxygalactosylceramide compound.Suitable cations include sodium ion, potassium ion, magnesium ion,calcium ion, and an ammonium cation such as tetramethylammonium ion.Examples of prodrugs include esters and other pharmaceuticallyacceptable derivatives, which, upon administration to a subject, arecapable of providing active 6″-amino-6″-deoxygalactosylceramidecompounds.

The compounds of this invention may be modified by appending appropriatefunctionalities to enhance selective biological properties. Suchmodifications are known in the art and include those which increasebiological penetration into a given biological compartment (e.g., blood,lymphatic system, central nervous system), increase oral availability,increase solubility to allow administration by injection, altermetabolism and alter rate of excretion.

Methods of Using 6″-amino-6″-deoxygalactosylceramide compounds

The effect of a particular C₆″-substitution on a6″-amino-6″-deoxygalactosylceramide compound's ability to stimulate NKTcells can be evaluated e.g., by measuring interleukin (IL)-2 productionusing an immobilized CD1d assay (Benlagha, K.; Weiss, A.; Beavis, A.;Teyton, L.; Bendalac, A. J. Exp. Med. 2000, 191, 1895). The assayincludes loading soluble, biotinylated CD1d onto precoated avidinplates, pulsing the plates with incrementally varied concentrations ofglycolipids, washing the plates, treating the plates with aCD1d-restricted Vα24 NKT cell hybridoma, and measuring IL-2 releaseusing ELISA (enzyme-linked immunosorbent assay).

Each compound's NKT cell stimulating ability is compared against that ofthe reference compound, KRN7000, 14 (Morita, M., et al., J. Med. Chem.1995, 38, 2176).

Compounds 8, 10, 12 and 13 were determined to possess relatively highNKT cell stimulating abilities according to the above assay. The resultsin FIGS. 1 and 2 indicate that there is a dose-dependent response to theglycolipids (i.e., cytokine release) and that this response iscomparable to that of the reference compound. Although 8 and 12 appearslightly less efficient in the results from the experiment shown in FIG.1, no significant differences among the compounds were found in repeatedexperiments (at least three experiments for each compound). In aseparate series of experiments (e.g., FIG. 2), compound 13 was slightly,but reproducibly more efficient in stimulating NKT cells than 1. Similarresults were observed using CD1d transfected rat basophilic leukemiacells for antigen presentation to NKT cell hybridomas.

The attachment of a dansyl group directly at C₆″ (as in 8) or through afive carbon tether (as in 10) did not cause a significant loss ofstimulating properties. Similarly, alteration of the appended group(i.e., dansyl vs, prodan vs. biotin) did not greatly affect theabilities of these glycolipids to stimulate NKT cells.

Binding of the glycolipids with CD1d and NKT cell receptors can bevisualized by fluorescence modulation studies, fluorescence and surfaceplasmon resonance studies, which employ6″-amino-6″-deoxygalactosylceramide probe compounds that contain one ormore reporter groups attached directly or indirectly to the 6″-aminogroup (e.g., probe compounds described herein). In certain embodiments,probes that contain fluorophores as the reporting group can be used toquantify the association between the glycolipids and CD1d and the NKTcell receptors during NKT cell stimulation. The selection ofexperimental protocols to observe association are within the art and aredescribed in Kasten, F. H., “Introduction to Fluorescent Probes:Properties, History and Applications” in Fluorescent and LuminescentProbes for Biological Activit, W. T. Mason, Ed., Academic Press (1993)pp. 12-33 and Lakowicz, J. R., Ed., Topics in Fluorescence Spectroscopy:Probe Design and Chemical Sensing (Volume 4), Plenum Publishing (1994).

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate,dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate,glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts. Salts derived from appropriate bases include alkalimetal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammoniumand N-(alkyl)₄ ⁺ salts. This invention also envisions the quaternizationof any basic nitrogen-containing groups of the compounds disclosedherein. Water or oil-soluble or dispersible products may be obtained bysuch quaternization. Salt forms of the compounds of any of the formulaeherein can be amino acid salts of carboxy groups (e.g. L-arginine,-lysine, -histidine salts).

The compounds of the formulae described herein can, for example, beadministered by injection, intravenously, intraarterially, subdermally,intraperitoneally, intramuscularly, or subcutaneously; or orally,buccally, nasally, transmucosally, topically, in an ophthalmicpreparation, or by inhalation, with a dosage ranging from about 0.5 toabout 100 mg/kg of body weight, alternatively dosages between 1 mg and1000 mg/dose, every 4 to 120 hours, or according to the requirements ofthe particular drug. The methods herein contemplate administration of aneffective amount of compound or compound composition to achieve thedesired or stated effect. Typically, the pharmaceutical compositions ofthis invention will be administered from about 1 to about 6 times perday or alternatively, as a continuous infusion. Such administration canbe used as a chronic or acute therapy. The amount of active ingredientthat may be combined with the carrier materials to produce a singledosage form will vary depending upon the host treated and the particularmode of administration. A typical preparation will contain from about 5%to about 95% active compound (w/w). Alternatively, such preparationscontain from about 20% to about 80% active compound.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level. Patients may, however,require intermittent treatment on a long-term basis upon any recurrenceof disease symptoms.

The compositions delineated herein include the compounds of the formulaedelineated herein, as well as additional therapeutic agents if present,in amounts effective for achieving a modulation of disease or diseasesymptoms, including those described herein.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompounds of the formulae described herein.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir, preferably by oraladministration or administration by injection. The pharmaceuticalcompositions of this invention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agentswhich are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and or suspensions. Othercommonly used surfactants such as Tweens or Spans and/or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.If desired, certain sweetening and/or flavoring and/or coloring agentsmay be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisinvention is useful when the desired treatment involves areas or organsreadily accessible by topical application. For application topically tothe skin, the pharmaceutical composition should be formulated with asuitable ointment containing the active components suspended ordissolved in a carrier. Carriers for topical administration of thecompounds of this invention include, but are not limited to, mineraloil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier with suitable emulsifying agents. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water. The pharmaceuticalcompositions of this invention may also be topically applied to thelower intestinal tract by rectal suppository formulation or in asuitable enema formulation. Topically-transdermal patches are alsoincluded in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

A composition having the compound of the formulae herein and anadditional agent (e.g., a therapeutic agent) can be administered usingan implantable device. Implantable devices and related technology areknown in the art and are useful as delivery systems where a continuous,or timed-release delivery of compounds or compositions delineated hereinis desired. Additionally, the implantable device delivery system isuseful for targeting specific points of compound or composition delivery(e.g., localized sites, organs). Negrin et al., Biomaterials, 22(6):563(2001). Timed-release technology involving alternate delivery methodscan also be used in this invention. For example, timed-releaseformulations based on polymer technologies, sustained-release techniquesand encapsulation techniques (e.g., polymeric, liposomal) can also beused for delivery of the compounds and compositions delineated herein.

Also within the invention is a patch to deliver active chemotherapeuticcombinations herein. A patch includes a material layer (e.g., polymeric,cloth, gauze, bandage) and the compound of the formulae herein asdelineated herein. One side of the material layer can have a protectivelayer adhered to it to resist passage of the compounds or compositions.The patch can additionally include an adhesive to hold the patch inplace on a subject. An adhesive is a composition, including those ofeither natural or synthetic origin, that when contacted with the skin ofa subject, temporarily adheres to the skin. It can be water resistant.The adhesive can be placed on the patch to hold it in contact with theskin of the subject for an extended period of time. The adhesive can bemade of a tackiness, or adhesive strength, such that it holds the devicein place subject to incidental contact, however, upon an affirmative act(e.g., ripping, peeling, or other intentional removal) the adhesivegives way to the external pressure placed on the device or the adhesiveitself, and allows for breaking of the adhesion contact. The adhesivecan be pressure sensitive, that is, it can allow for positioning of theadhesive (and the device to be adhered to the skin) against the skin bythe application of pressure (e.g., pushing, rubbing) on the adhesive ordevice.

When the compositions of this invention comprise a combination of acompound of the formulae described herein and one or more additionaltherapeutic or prophylactic agents, both the compound and the additionalagent should be present at dosage levels of between about 1 to 100%, andmore preferably between about 5 to 95% of the dosage normallyadministered in a monotherapy regimen. The additional agents may beadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents may be part ofa single dosage form, mixed together with the compounds of thisinvention in a single composition.

The compounds of the invention can be used in the treatment of cancer.The cancer can be, but is not limited to: a human leukemia, sarcoma,osteosarcoma, lymphoma, melanoma, ovarian, skin, testicular, gastric,pancreatic, renal, breast, prostate colorectal, head and neck, brain,esophageal, bladder, adrenal cortical, lung, bronchus, endometrial,cervical or hepatic cancer, or cancer of unknown primary site.

The compounds of the invention can also be used in the treatment of anautoimmune diseases. The autoimmune disease can be, but is not limitedto: (1) a rheumatic disease such as rheumatoid arthritis, systemic lupuserythematosus, Sjögren's syndrome, scleroderma, mixed connective tissuedisease, dermatomyositis, polymyositis, Reiter's syndrome or Behcet'sdisease (2) type I or type II diabetes (3) an autoimmune disease of thethyroid, such as Hashimoto's thyroiditis or Graves' Disease (4) anautoimmune disease of the central nervous system, such as multiplesclerosis, myasthenia gravis, or encephalomyelitis (5) a variety ofphemphigus, such as phemphigus vulgaris, phemphigus vegetans, phemphigusfoliaceus, Senear-Usher syndrome, or Brazilian phemphigus, (6) diseasesof the skin such as psoriasis or neurodermitis, and (7) inflammatorybowel disease (e.g., ulcerative colitis or Crohn's Disease).

The invention will be further described in the following examples. Itshould be understood that these examples are for illustrative purposesonly and are not to be construed as limiting this invention in anymanner.

Example 1 Preparation of6-azido-2,3,4-tetra-O-benzyl-6-deoxymethylgalactopyranoside (4)

Compound 3 (1.86 g, 6.52 mmol) was dissolved in MeOH (20 mL), cooled to0° C., and acetyl chloride (4.35 mL) was added. The mixture was allowedwarm to room temperature and stirred for 12 h. The solvent was removedin vacuo, and the residue was chromatographed (SiO₂, 10% MeOH in CH₂Cl₂)to afford 6-azido-6-deoxymethylgalactopyranoside (mixture of anomers) asa white solid (1.23 g, 86% yield). ¹H NMR (10% CD₃OD in CDCl₃) δ 4.79(d, J=2.5 Hz, 1H), 4.46 (br, 1H), 3.92 (dd, J=8.5, 4.0 Hz, 1H),3.84-3.75 (m, 3H), 3.63 (dd, J=12.5, 8.5 Hz, 1H), 3.46 (s, 3H), 3.31(dd, J=13.0, 4.5 Hz, 1H); ¹³C NMR (10% CD₃OD in CDCl₃) δ 99.80, 69.84,69.66, 69.54, 68.55, 55.08, 51.21; HRFAB-MS (thioglycerol+H⁺ matrix) m/e([M+H]⁺) 220.0951 (3.1%), calcd 220.0933.

To a mixture of 6-azido-6-deoxymethylgalactopyranoside (482 mg, 2.2mmol) in THF (30 mL) was added benzyl bromide (1.57 mL, 13.2 mmol),K₂CO₃ (2.4 g, 17.6 mmol) and 18-crown-6 (120 mg). The suspension wasstirred for 15 min, and NaH (0.396 g, 60% in mineral oil, 16.5 mmol) wasadded. After 12 h, brine (30 mL) was added and the product was extractedwith 10% EtOAc in hexane (3×20 mL). The combined extracts were driedover Na₂SO₄ and concentrated in vacuo. The desired product 4 (1.02 g,95% yield) was obtained as a clear oil after chromatography (SiO₂,EtOAc:hexanes 1:2). NMR (¹H, CDCl₃) δ 7.40-7.25 (m, 15H), 5.02-4.62 (m,7H), 4.14-3.76 (m, 4H), 3.57-3.48 (m, 1H), 3.39 (s, 3H), 2.94 (dd,J=12.4, 4.4 Hz, 1H); NMR (¹³C, CDCl₃) δ 138.65, 138.58, 138.34, 128.68,128.61, 128.32, 128.12, 128.01, 127.87, 127.78, 99.01, 79.16, 76.48,75.45, 74.81, 73.89, 69.98, 55.71, 51.64; HRFAB-MS (thioglycerol+H⁺matrix) m/e ([M+H]⁺) 490.2347 (3.6%), calcd 490.2342.

Example 2 Preparation of6-azido-2,3,4-tetra-O-benzyl-6-deoxy-α-galactosyl fluoride (5)

Acetic anhydride (0.45 mL) was added to a solution of 4 (398 mg, 0.81mmol) in acetic acid (0.33 mL). The mixture was cooled to 0° C., andconcentrated H₂SO₄ (6.8 μl) was added. The mixture was stirred at 0° C.for 8 h, and H₂O (5 mL) was added. The product was extracted with CH₂Cl₂(3×5 mL), and the combined extracts were dried over Na₂SO₄ andconcentrated in vacuo. After chromatography (SiO₂, EtOAc:hexanes 1:2),6-azido-2,3,4-tetra-O-benzyl-6-deoxy-α-galactosyl 1-acetate (354 mg, 84%yield) was obtained as a clear oil. NMR (¹H, CDCl₃) δ 7.39-7.28 (m,15H), 6.38 (d, J=3.5 Hz, 1H), 5.02-4.58 (m, 6H), 4.17 (dd, J=11.0, 4.0Hz, 1H), 3.91-3.88 (m, 3H), 3.47 (dd, J=12.5, 7.0 Hz, 1H), 3.15 (dd,J=12.5, 7.0 Hz, 1H), 2.12 (s, 3H); NMR (¹³C, CDCl₃) δ 169.55, 138.59,138.13, 138.00, 128.68, 128.62, 128.57, 128.56, 128.53, 128.51, 128.18,128.13, 128.10, 128.03, 127.98, 127.85, 127.79, 127.60, 90.65, 78.67,75.45, 75.31, 74.95, 74.69, 74.60, 74.42, 73.57, 73.53, 71.89, 50.85,21.28; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e ([M+Na]⁺) 540.2112 (100%),calcd 540.2111.

Anhydrous pyridine (0.6 mL) and 70% hydrogen fluoride-pyridine (1.5 mL)were placed in a 50-ml polyethylene vessel. To this mixture cooled to−20° C. was added a solution of6-azido-2,3,4-tetra-O-benzyl-6-deoxy-α-galactosyl 1-acetate (401 mg,0.77 mmol) in toluene (0.3 mL). The mixture was allowed to warm to 0° C.and stir for 6 h then poured into a mixture of ether (10 mL) andsaturated aqueous potassium fluoride (30 mL). The product was extractedwith a 3:1 ether-hexane solution (2×50 mL), and the combined extractswere washed with saturated aqueous potassium fluoride (30 mL) and brine(30 mL). The organics were dried over Na₂SO₄ and the solvent was removedin vacuo. The residue was purified chromatographically (SiO₂,EtOAc:hexanes 1:2) to give6-azido-2,3,4-tetra-O-benzyl-6-deoxy-α-galactosyl fluoride as a clearoil (230 mg, 78% yield). NMR (¹H, CDCl₃) δ 7.40-7.25 (m, 15H), 5.63 (dd,J=54.0, 2.5 Hz, 1H), 5.00 (d, J=11.5 Hz, 1H), 4.88-4.72 (m, 4H), 4.61(d, J=11.0 Hz, 1H), 4.01-3.88 (m, 4H), 3.51 (dd, J=12.5, 7.5 Hz, 1H),3.13 (dd, J=12.0, 6.0 Hz, 1H); NMR (¹³C, CDCl₃) δ 138.38, 138.09,138.07, 128.74, 128.71, 128.66, 128.56, 128.23, 128.20, 128.16, 128.04,127.80, 107.12, 105.32, 78.45, 75.85, 75.67, 74.99, 74.48, 73.99, 73.67,72.14, 72.11, 50.96; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e ([M+Na]⁺)500.1956 (100%), calcd 500.1962.

Example 3 Preparation of Compound 7

To a solution of(2S,3S,4R)-3,4,-bis-t-butyldimethylsilyloxy-2-hexacosanoylamino-4-octadecanol(6) (266 mg, 0.28 mmol) in THF (10 mL), SnCl₂ (163.7 mg, 0.86 mmol),AgClO₄ (179 mg, 0.86 mmol) and powdered 4 Å molecular sieves (1.34 g)were added. A solution of 5 (214 mg, 0.45 mmol) in THF (2 mL) was thenadded at −10° C. The reaction mixture was allowed to warm gradually toroom temperature with stirring over the course of 2 h. The mixture wasfiltered through Celite, and the filter cake was washed with Et₂O. Thecombined filtrate was concentrated under reduced pressure. The residuewas purified chromatographically (SiO₂, EtOAc:hexanes 1:2) to givecompound 7 (175 mg, 44% yield) as a clear oil. NMR (¹H, CDCl₃) δ7.40-7.31 (m, 15H), 5.92 (d, J=8.0 Hz, 1H), 5.02 (d, J=11.0 Hz, 1H),4.85-4.59 (m, 6H), 4.21 (m, 1H), 4.06-3.99 (m, 2H), 3.91 (dd, J=13.0,3.0 Hz, 1H), 3.86-3.83 (m, 3H), 3.79 (m, 1H), 3.50 (dd, J=12.0, 7.5 Hz,1H), 3.17 (dd, J=12.0, 7.5 Hz, 1H), 2.03 (t, J=7.5 Hz, 2H), 1.58-1.24(m, 73H), 0.92 (s, 9H), 0.91 (s, 9H), 0.89 (m, 6H), 0.09 (s, 3H), 0.08(s, 3H), 0.06 (s, 3H), 0.05 (s, 3H); NMR (¹³C, CDCl₃) δ 173.24, 138.69,138.61, 138.35, 128.64, 128.58, 128.57, 128.06, 128.03, 127.94, 127.85,127.64, 100.18, 79.16, 76.59, 76.20, 75.99, 75.04, 74.87, 73.71, 73.49,70.11, 69.56, 51.78, 51.38, 37.05, 33.59, 32.14, 32.13, 30.10, 29.93,29.91, 29.89, 29.87, 29.81, 29.77, 29.68, 29.66, 29.58, 26.31, 26.24,25.86, 22.90, 18.52, 18.37, 14.34, −3.471, −3.756, −4.442, −4.705;HRFAB-MS (thioglycerol+H⁺ matrix) m/e ([M+H]⁺) 1382.0592 (81.3%), calcd1382.0601.

Example 4 Preparation of Compound 2

To a solution of 7 (175 mg, 0.12 mmol) in THF (4 mL), TBAF (1.0 M inTHF, 0.5 mL, 0.5 mmol) was added dropwise at room temperature. Afterstirring for 1.5 h, the mixture was diluted with water and extractedwith Et₂O. The extract was washed with water and brine, dried (MgSO₄),and concentrated under reduced pressure. The residue was purified bycolumn chromatography (SiO₂, EtOAc:hexanes 1:2) to give thecorresponding diol (118 mg, 81% yield) as a clear glass. NMR (¹H, CDCl₃)δ 7.39-7.25 (m, 15H), 6.25 (d, J=8.0 Hz, 1H), 4.99 (d, J=11.0 Hz, 1H),4.88-4.57 (m, 6H), 4.27 (m, 1H), 4.05 (dd, J=9.5, 3.0 Hz, 1H), 3.93 (dd,J=10.0, 3.0 Hz, 1H), 3.87-3.80 (m, 2H), 3.72 (m, 1H), 3.51-3.45 (m, 3H),3.03 (dd, J=13.0, 6.0 Hz, 1H), 2.25 (d, J=5.5 Hz, 1H), 2.15 (t, J=7.0Hz, 2H), 2.13-1.25 (m, 76H), 0.88 (t, J=7.5 Hz, 6H); NMR (¹³C, CDCl₃) δ173.07, 138.30, 138.12, 137.84, 128.75, 128.74, 128.64, 128.58, 128.32,128.29, 128.20, 128.02, 127.72, 99.08, 79.38, 76.41, 75.93, 74.84,74.60, 74.49, 73.54, 73.27, 70.42, 69.99, 51.22, 49.28, 37.03, 33.58,32.13, 29.94, 29.91, 29.88, 29.75, 29.63, 29.57, 29.52, 26.12, 25.98,22.90, 14.33; HRFAB-MS (thioglycerol+H⁺ matrix) m/e ([M+H]⁺) 1151.9072(89.4%), calcd 1151.9079.

To a solution of the diol (118 mg, 0.1 mmol) in THF/H₂O (1.5 mL/0.3 mL)was added triphenylphosphine (40.3 mg). The reaction mixture was stirredat room temperature for 12 h. The resulting amine (114 mg, 99% yield)was obtained as a clear glass after chromatography (SiO₂,CHCl₃:MeOH:NH₃—H₂O 1:0.4:0.02). NMR (¹H, CDCl₃) δ 7.59-7.18 (m, 15H),6.49 (d, J=8.5 Hz, 1H), 4.87 (d, J=11.5 Hz, 1H), 4.78-4.52 (m, 6H), 4.18(m, 1H), 3.97 (dd, J=10.0, 3.5 Hz, 1H), 3.81 (dd, J=10.0, 4.5 Hz, 1H),3.77 (dd, J=10.0, 2.5 Hz, 1H), 3.72 (m, 2H), 3.67 (dd, J=10.0, 4.0 Hz,1H), 3.52 (dd, J=8.0, 5.0 Hz, 1H), 3.41 (m, 2H), 2.82 (dd, J=13.0, 8.0Hz, 1H), 2.42 (dd, J=13.0, 5.0 Hz, 1H), 2.37 (m, 1H), 2.04 (t, J=8.0 Hz,2H), 1.49-0.82 (m, 74H), 0.79 (t, J=7.0 Hz, 6H); NMR (¹³C, CDCl₃) δ173.54, 138.60, 138.33, 138.12, 132.96, 132.31, 132.23, 132.19, 132.17,132.14, 128.76, 128.74, 128.66, 128.59, 128.24, 128.11, 128.09, 127.86,127.69, 98.86, 79.81, 76.50, 76.35, 74.79, 74.62, 74.08, 73.37, 73.03,72.58, 68.51, 53.93, 50.42, 42.43, 36.97, 33.88, 32.11, 29.96, 29.90,29.84, 29.74, 29.61, 29.55, 29.52, 26.16, 25.97, 25.86, 22.88, 20.92,14.32; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e ([M+Na]⁺) 1149.8790(100%), calcd 1149.8786.

To liquid NH₃ (ca. 8 mL) under N₂ at −78° C. was added Na° (20 mg), andthe mixture was stirred for 2 min. To the blue solution was added theamine (18 mg, 0.016 mmol) in THF (1 mL), and the mixture was stirred for40 min at −78° C. The reaction was quenched by addition of MeOH (4 mL).Ammonia was removed with a stream of N₂, and the solution was dilutedwith MeOH to 8 mL. The solution was concentrated under reduced pressure,and the residue was purified by column (SiO₂, CHCl₃:MeOH:NH₃—H₂O1:0.4:0.02) to give 2 (7.3 mg, 53%) as a white solid. NMR (¹H, 5% CD₃ODin CDCl₃) δ 4.91 (d, J=4.0 Hz, 1H), 4.21 (m, 1H), 3.88 (m, 2H), 3.80(dd, J=10.0, 3.5 Hz, 1H), 3.75 (m, 1H), 3.70 (dd, J=10.0, 3.5 Hz, 1H),3.62-3.51 (m, 10H), 3.06 (dd, J=13.0, 7.5 Hz, 1H), 2.90 (dd, J=13.0, 4.0Hz, 1H), 2.19 (t, J=8.0 Hz, 2H), 1.68-1.25 (m, 73H). 0.88 (t, J=7.0 Hz,6H); NMR (¹³C, 5% CD₃OD in CDCl₃) δ 174.36, 99.75, 75.17, 72.06, 70.84,70.37, 70.22, 68.92, 67.31, 50.36, 42.40, 36.62, 33.02, 31.97, 29.83,29.77, 29.74, 29.71, 29.70, 29.61, 29.46, 29.42, 25.90, 25.87, 22.73,14.11; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e ([M+Na]⁺) 879.7384 (100%),calcd 879.7377.

Example 5 Preparation of Compound 8

Dansyl chloride (1.5 mg, 0.0055 mmol) was added to a solution of 2 (4.5mg, 0.005 mmol) in pyridine (1 mL), and the mixture was stirred for 5 h.The pyridine was removed in vacuo, and the product was purifiedchromatographically (SiO₂, 10% MeOH in CH₂Cl₂) giving a light yellowglass (3.4 mg, 60% yield). NMR (¹H, 5% CD₃OD in CDCl₃) δ 8.55 (d, J=9.0Hz, 1H), 8.26 (d, J=8.5 Hz, 1H), 8.19 (dd, J=7.0, 1.5 Hz, 1H), 7.58-7.50(m, 2H), 7.20 (d, J=7.5 Hz, 1H), 6.93 (d, J=8.5 Hz, 1H), 4.83 (d, J=2.5Hz, 1H), 4.22 (m, 1H), 3.91 (m, 3H), 3.72 (m, 2H), 3.64-3.55 (m, 4H),3.07 (m, 2H), 2.88 (s, 6H), 2.22 (br, 7H), 1.62-1.25 (m, 72H), 0.87 (t,J=7.0 Hz, 6H); NMR (¹³C, 5% CD₃OD in CDCl₃) δ 174.45, 152.09, 134.62,130.69, 130.10, 129.71, 129.36, 123.31, 118.91, 115.43, 99.68, 75.36,72.41, 70.18, 69.55, 69.13, 69.03, 67.96, 50.42, 45.56, 43.28, 36.78,33.14, 32.07, 29.93, 29.88, 29.85, 29.82, 29.80, 29.72, 29.57, 29.52,25.96, 25.94, 22.84, 14.25; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e([M+Na]⁺) 1112.7867 (100%), calcd 1112.7887.

Example 6 Preparation of Compound 10

Ester 9 (9.4 mg, 0.02 mmol) was added to a solution of 2 (5.1 mg, 0.0059mmol) in pyridine (1 mL), and the mixture was stirred for 12 h. Thepyridine was removed in vacuo, and the product was purified by columnchromatography (SiO₂, 10% MeOH in CH₂Cl₂) giving a light yellow glass(3.7 mg, 53% yield). NMR (¹H, 5% CD₃OD in CDCl₃) δ 8.53 (d, J=9.0 Hz,1H), 8.28 (d, J=8.5 Hz, 1H), 8.20 (dd, J=7.5, 1.2 Hz, 1H), 7.59-7.50 (m,2H), 7.20 (d, J=7.5 Hz, 1H), 7.04 (d, J=8.5 Hz, 1H), 4.91 (d, J=3.0 Hz,1H), 4.17 (m, 1H), 3.90 (dd, J=10.5, 4.5 Hz, 1H), 3.84 (m, 4H), 3.75(dd, J=11.0, 4.5 Hz, 1H), 3.68-3.64 (m, 2H), 3.57-3.54 (m, 2H), 3.20(dd, J=13.5, 5.5 Hz, 1H), 2.89 (s, 6H), 2.80 (m, 1H), 2.46-2.03 (m,11H), 1.62-1.25 (m, 78H), 0.87 (t, J=7.5 Hz, 6H); NMR (¹³C, 5% CD₃OD inCDCl₃) δ 175.38, 174.50, 152.04, 134.69, 130.52, 130.01, 129.72, 129.61,128.46, 123.31, 118.98, 115.40, 99.71, 74.97, 72.45, 69.94, 69.01,68.95, 68.03, 50.39, 45.54, 42.76, 42.61, 39.45, 36.57, 35.87, 32.79,32.06, 29.85, 29.68, 29.50, 29.41, 28.97, 26.85, 26.00, 25.92, 25.49,25.03, 24.77, 24.37, 22.82, 14.23; HRFAB-MS (thioglycerol+Na⁺ matrix)m/e ([M+Na]⁺) 1225.8741 (100%), calcd 1225.8728.

Example 7 Preparation of Compound 12

Ester 11 (6.6 mg, 0.018 mmol) was added to a solution of 2 (5.0 mg,0.0058 mmol) in pyridine (1 mL), and the mixture was stirred for 12 h.The pyridine was removed in vacuo, and the product was purified bycolumn chromatography (SiO₂, 10% MeOH in CH₂Cl₂) giving compound 12 as aclear glass (3.0 mg, 46% yield). NMR (¹H, 5% CD₃OD in CDCl₃) δ 8.36 (m,1H), 7.89 (dd, J=8.0, 1.5 Hz, 1H), 7.81 (d, J=8.5 Hz, 1H), 7.64 (d,J=8.5 Hz, 1H), 7.18 (dd, J=8.5, 2.5 Hz, 1H), 7.01 (d, J=8.0 Hz, 1H),6.86 (d, J=2.5 Hz, 1H), 4.89 (d, J=4.0 Hz, 1H), 4.19 (m, 1H), 3.92 (dd,J=10.0, 4.5 Hz, 1H), 3.82-3.38 (m, 9H), 3.22 (dd, J=13.5, 6.0 Hz, 1H),3.19 (s, 6H), 2.65 (t, J=7.0 Hz, 2H), 2.34 (br, 5H), 2.18 (m, 2H),2.05-2.08 (m, 2H), 1.65-1.23 (m, 72H), 0.87 (t, J=7.0 Hz, 6H); NMR (¹³C,5% CD₃OD in CDCl₃) δ 199.01, 174.46, 150.56, 138.06, 130.98, 130.42,129.86, 126.43, 125.10, 124.35, 121.72, 116.49, 110.59, 105.36, 99.72,84.07, 75.29, 72.47, 70.11, 69.23, 68.99, 68.69, 67.80, 60.06, 50.56,40.54, 36.76, 33.62, 33.11, 32.07, 30.23, 29.86, 29.85, 29.81, 29.80,29.71, 29.57, 29.51, 25.99, 22.83, 14.25; HRFAB-MS (thioglycerol+Na⁺matrix) m/e ([M+Na]⁺) 1132.8484 (100%), calcd 1132.8480.

Example 8 Preparation of Compound 13

N-hydroxysuccinimidobiotin (5.9 mg, 0.017 mmol) and Et₃N (30 ul) wereadded to a solution of 2 (5.0 mg, 0.0058 mmol) in DMF (1.5 mL). Themixture was stirred for 12 h, and applied directly to an SiO₂ column.Elution with 10% MeOH in CH₂Cl₂ gave the product 13 as a clear glass(3.2 mg, 52% yield). NMR (¹H, Pyridine-d₅) δ 8.86 (m, 1H), 8.61 (d,J=9.0 Hz, 1H), 7.54-7.40 (m, 4H), 5.54 (d, J=4.0 Hz, 1H), 5.28 (br, OH),4.66-4.61 (m, 2H), 4.56-4.50 (m, 3H), 4.41-4.31 (m, 6H), 4.23 (m, 1H),3.92 (m, 1H), 3.27 (m, 1H), 3.16 (m, 1H), 3.0-2.85 (m, 10H), 2.55-2.44(m, 6H), 2.32 (m, 1H), 1.96-1.50 (m, 20H), 1.32-1.26 (m, 44H), 0.87 (t,J=7.0 Hz, 6H); NMR (¹³C, pyridine-d₅) δ 170.36, 169.46, 164.28, 101.41,76.88, 72.57, 71.30, 71.21, 70.74, 70.10, 68.62, 62.41, 62.36, 60.57,56.30, 56.13, 51.24, 41.22, 41.15, 36.86, 36.27, 34.52, 32.17, 30.89,30.46, 30.23, 30.11, 29.98, 29.89, 29.85, 29.67, 29.12, 28.87, 28.53,26.56, 26.50, 26.27, 26.16, 26.10, 24.85, 22.98, 14.33; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e ([M+Na]⁺) 1105.8143 (100%), calcd1105.8153.

Example 9 In Vivo Antitumor Assay

Experiment is performed with groups consisting of 6 female BDF₁ mice, 6weeks old, B16 mouse melanoma cells (1×10⁶) are inoculatedsubcutaneously in the rear part of mice (day 0). On 1, 5, and 9 daysafter inoculation, a sample in a level of 0.1 mg/kg is administered tothe tail vein in a dose of 0.2 ml/20 g/mouse. The volume of tumor in thesubcutaneous rear partis measured on 8, 12, 16, and 20 days to determinethe tumor growth inhibiting rate of each sample.

Example 10 In Vivo Antiautoimmune Activity Assay

Representative compounds of the formulae herein are screened forantiautoimmune activity in a nonobese diabetic (NOD) mouse assayessentially as described in Wang, B.; Geng, Y.-B.; Wang, C.-R. J. Exp.Med. 2001, 194, 313-319, incorporated herein by reference.

Example 11 In Vivo Antiautoimmune Activity Assay

Representative compounds of the formulae herein are screened forantiautoimmune activity in an experimental autoimmune encephalomyelitis(EAE) assay essentially as described in Pal, E.; Tabira, T.; Kawano, T.;Taniguchi, M.; Miyake, S.; Yamamamura, T. J. Immunol. 2001, 166,662-668, incorporated herein by reference.

All references cited herein, whether in print, electronic, computerreadable storage media or other form, are expressly incorporated byreference in their entirety, including but not limited to, abstracts,articles, journals, publications, texts, treatises, internet web sites,databases, patents, and patent publications.

Other embodiments are in the claims.

1. A compound of Formula (I):

wherein, R₁ is: (i) hydrogen; or (ii) —SO₂R₁₀, wherein R₁₀ is: halo;hydroxy; OR₁₁; OR₁₂; amino; NHR₁₁; N(R₁₁)₂; NHR₁₂; N(R₁₂)₂;aralkylamino; or C₁-C₁₂ alkyl optionally substituted with halo, hydroxy,oxo, nitro, OR₁₁, OR₁₂, acyloxy, amino, NHR₁₁; N(R₁₁)₂; NHR₁₂; N(R₁₂)₂,aralkylamino, mercapto, thioalkoxy, S(O)R₁₁, S(O)R₁₂, SO₂R₁₁, SO₂R₁₂,NHSO₂R₁₁, NHSO₂R₁₂, sulfate, phosphate, cyano, carboxyl, C(O)R₁₁,C(O)R₁₂, C(O)OR₁₁, C(O)NH₂, C(O)NHR₁₁, C(O)N(R₁₁)₂, C₃-C₁₀ cycloalkylcontaining 0-3 R₁₃, C₃-C₁₀ heterocyclyl containing 0-3 R₁₃, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₅-C₁₀ cycloalkenyl, C₅-C₁₀ heterocycloalkenyl,C₆-C₂₀ aryl containing 0-3 R₁₄, or heteroaryl containing 0-3 R₁₄; orC₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocyclyl, C₅-C₁₀ cycloalkenyl, or C₅-C₁₀heterocycloalkenyl optionally substituted with one or more halo,hydroxy, oxo, OR₁₁, OR₁₂, acyloxy, nitro, amino, NHR₁₁, N(R₁₁)₂, NHR₁₂,N(R₁₂)₂, aralkylamino, mercapto, thioalkoxy, S(O)R₁₁, S(O)R₁₂, SO₂R₁₁,SO₂R₁₂, NHSO₂R₁₁, NHSO₂R₁₂, sulfate, phosphate, cyano, carboxyl,C(O)R₁₁, C(O)R₁₂, C(O)OR₁₁, C(O)NH₂, C(O)NHR₁₁, C(O)N(R₁₁)₂, alkyl,haloalkyl, C₃-C₁₀ cycloalkyl containing 0-3 R₁₃, C₃-C₁₀ heterocyclylcontaining 0-3 R₁₃, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₅-C₁₀ cycloalkenyl,C₅-C₁₀ heterocycloalkenyl, C₆-C₂₀ aryl heteroaryl containing 0-3 R₁₄, orC₆-C₂₀ heteroaryl containing 0-3 R₁₄; or C₂-C₆ alkenyl, C₂-C₆ alkynyl,aryl, or heteroaryl optionally substituted with one or more halo,hydroxy, OR₁₁, OR₁₂, acyloxy, nitro, amino, NHR₁₁, N(R₁₁)₂, NHR₁₂,N(R₁₂)₂, aralkylamino, mercapto, thioalkoxy, S(O)R₁₁, S(O)R₁₂, SO₂R₁₁,SO₂R₁₂, NHSO₂R₁₁, NHSO₂R₁₂, sulfate, phosphate, cyano, carboxyl,C(O)R₁₁, C(O)R₁₂, C(O)OR₁₁, C(O)NH₂, C(O)NHR₁₁, C(O)N(R₁₁)₂, alkyl,haloalkyl, C₃-C₁₀ cycloalkyl containing 0-3 R₁₃, C₃-C₁₀ heterocyclylcontaining 0-3 R₁₃, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₅-C₁₀ cycloalkenyl,C₅-C₁₀ heterocycloalkenyl, C₆-C₂₀ aryl containing 0-3 R₁₄, or C₆-C₂₀heteroaryl containing 0-3 R₁₄; or (iii) —C(O)R₁₀, wherein R₁₀ is definedas above; or (iv) —C(R₁₀)₂(R₁₅), wherein R₁₀ is defined as above; R₁₅ ishydrogen, R₁₀, or R₁₅ and R₂ when taken together form a double bondbetween the carbon to which R₁₅ is attached and the nitrogen to which R₂is attached; or (v) R₁ and R₂ form a heterocyclyl of 3-10 ring atomsoptionally substituted with R₁₀; R₂ is hydrogen, or R₂ and R₁₅ whentaken together form a double bond between the carbon to which R₁₅ isattached and the nitrogen to which R₂ is attached, or R₂ and R₁ form aheterocyclyl of 3-10 ring atoms optionally substituted with R₁₀; R₃, R₄,R₅, R₆ and R₇ are each independently hydrogen, C₁-C₆ alkyl, C₆-C₁₂aralkyl, or C₁-C₆ acyl; R₈ is —(CH₂)_(x)CH₃; R₉ is a linear or branchedC₃-C₁₀₀ alkyl; R₁₁ is C₁-C₂₀ alkyl optionally substituted with halo,hydroxy, alkoxy, amino, alkylamino, dialkylamino, sulfate, or phosphate;R₁₂ is aryl optionally substituted with halo, haloalkyl, hydroxy,alkoxy, nitro, amino, alkylamino, dialkylamino, sulfate, or phosphate;Each R₁₃ is independently halo, haloalkyl, hydroxy, alkoxy, oxo, amino,alkylamino, dialkylamino, sulfate, or phosphate; Each R₁₄ isindependently halo, haloalkyl, hydroxy, alkoxy, nitro, amino,alkylamino, dialkylamino, sulfate, or phosphate; and x is 1-100.
 2. Thecompound of claim 1 wherein x is 24 and R₉ is n-tetradecyl.
 3. Thecompound of claim 2 wherein R₁ is SO₂R₁₀.
 4. The compound of claim 3wherein R₁₀ is aryl substituted with N(R₁₁)₂;
 5. (canceled)
 6. Thecompound of claim 2 wherein R₁ is C(O)R₁₀.
 7. The compound of claim 6wherein R₁₀ is C₁-C₆ alkyl substituted with halo, hydroxy, oxo, nitro,OR₁₁, OR₁₂, acyloxy, amino, NHR₁₁, N(R₁₁)₂, NHR₁₂, N(R₁₂)₂,aralkylamino, mercapto, thioalkoxy, S(O)R₁₁, S(O)R₁₂, SO₂R₁₁, SO₂R₁₂,NHSO₂R₁₁, NHSO₂R₁₂, sulfate, phosphate, cyano, carboxyl, C(O)R₁₁,C(O)R₁₂, C(O)OR₁₁, C(O)NH₂, C(O)NHR₁₁, C(O)N(R₁₁)₂, C₃-C₁₀ cycloalkylcontaining 0-3 R₁₃, C₃-C₁₀ heterocyclyl containing 0-3 R₁₃, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₅-C₁₀ cycloalkenyl, C₅-C₁₀ heterocycloalkenyl,C₆-C₂₀ aryl containing 0-3 R₁₄, or C₆-C₂₀ heteroaryl containing 0-3 R₁₄;8. The compound of claim 7 wherein R₁₀ is C₁-C₆ alkyl substituted withNHSO₂R₁₂.
 9. (canceled)
 10. The compound of claim 7, wherein R₁₀ isalkyl substituted with C(O)R₁₂.
 11. (canceled)
 12. The compound of claim7 wherein R₁₀ is alkyl is substituted with C₅-C₁₀ heterocyclylcontaining 0-3 R₁₃.
 13. (canceled)
 14. A probe for observing glycolipidassociation with CD1d and NKT cell receptors during NKT cell stimulationcomprising a formula of Formula (II):

wherein: X is —SO₂—, —C(O)—, or absent; Y is a linker group; and Z is areporter group.
 15. A method of quantifying glycolipid association withCD1d and NKT cell receptors during NKT cell stimulation comprising: (i)contacting a compound of Formula (II) with a CD1d protein; (ii) allowingthe compound to associate with the CD1d protein; (iii) measuringfluorescence emitted by the compound during steps (i) and (ii) toprovide one or more pre-NKT cell contact fluorescence measurements; (iv)contacting the compound and CD1d protein with an NKT cell line; (v)measuring fluorescence emitted by the compound during step (iv) toprovide one or more NKT cell contact fluorescence measurements. 16.(canceled)
 17. The method of claim 15 further comprising the step ofcomparing the fluorescence measurements in steps (iii) and (v).
 18. Amethod of stimulating NKT cells comprising contacting an NKT cell with acompound of Formula (I) and a CD1 protein,
 19. (canceled)
 20. A methodof stimulating the immune system of a subject in need of suchstimulation, the method comprising administering an effective amount ofa compound according to claim
 1. 21. A method of treating an autoimmunedisease in a subject in need of such treatment, the method comprisingadministering an effective amount of a compound according to claim 1.22. The method of claim 20 wherein the subject is a human.
 23. Themethod of claim 21 wherein the subject is a human.
 24. A method ofmaking a compound of Formula (I) comprising: (i) converting a compoundof Formula (III) to a compound of Formula (IV):

and (ii) contacting a compound of Formula (IV) with R₁-LG to afford acompound of Formula (I), wherein: R₁ is: (i) —SO₂R₁₀, wherein R₁₀ is:halo; hydroxy; OR₁₁; OR₁₂; amino; NHR₁₁; N(R₁₁)₂; NHR₁₂; N(R₁₂)₂;aralkylamino; or C₁-C₁₂ alkyl optionally substituted with halo, hydroxy,oxo, nitro, OR₁₁, OR₁₂, acyloxy, amino, NHR₁₁, N(R₁₁)₂, NHR₁₂, N(R₁₂)₂,aralkylamino, mercapto, thioalkoxy, S(O)R₁₁, S(O)R₁₂, SO₂R₁₁, SO₂R₁₂,NHSO₂R₁₁, NHSO₂R₁₂, sulfate, phosphate, cyano, carboxyl, C(O)R₁₁,C(O)R₁₂, C(O)OR₁₁, C(O)NH₂, C(O)NHR₁₁, C(O)N(R₁₁)₂, C₃-C₁₀ cycloalkylcontaining 0-3 R₁₃, C₃-C₁₀ heterocyclyl containing 0-3 R₁₃, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₅-C₁₀ cycloalkenyl, C₅-C₁₀ heterocycloalkenyl,C₆-C₂₀ aryl containing 0-3 R₁₄, or C₆-C₂₀ heteroaryl containing 0-3 R₁₄;or C₃-C₁₀ cycloalkyl, C₃-C₁₀ heterocyclyl, C₅-C₁₀ cycloalkenyl, orC₅-C₁₀ heterocycloalkenyl optionally substituted with one or more halo,hydroxy, oxo, OR₁₁, OR₁₂, acyloxy, nitro, amino, NHR₁₁, N(R₁₁)₂, NHR₁₂,N(R₁₂)₂, aralkylamino, mercapto, thioalkoxy, S(O)R₁₁, S(O)R₁₂, SO₂R₁₁,SO₂R₁₂, NHSO₂R₁₁, NHSO₂R₁₂, sulfate, phosphate, cyano, carboxyl,C(O)R₁₁, C(O)R₁₂, C(O)OR₁₁, C(O)NH₂, C(O)NHR₁₁, C(O)N(R₁₁)₂, alkyl,haloalkyl, C₃-C₁₀ cycloalkyl containing 0-3 R₁₃, C₃-C₁₀ heterocyclylcontaining 0-3 R₁₃, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₅-C₁₀ cycloalkenyl,C₅-C₁₀ heterocycloalkenyl, C₆-C₂₀ aryl containing 0-3 R₁₄, or C₆-C₂₀heteroaryl containing 0-3 R₁₄; or C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, orheteroaryl optionally substituted with one or more halo, hydroxy, OR₁₁,OR₁₂, acyloxy, nitro, amino, NHR₁₁, N(R₁₁)₂, NHR₁₂, N(R₁₂)₂,aralkylamino, mercapto, thioalkoxy, S(O)R₁₁, S(O)R₁₂, SO₂R₁₁, SO₂R₁₂,NHSOR₁₁, NHSO₂R₁₂, sulfate, phosphate, cyano, carboxyl, C(O)R₁₁,C(O)R₁₂, C(O)OR₁₁, C(O)NH₂, C(O)NHR₁₁, C(O)N(R₁₁)₂, alkyl, halo alkyl,C₃-C₁₀ cycloalkyl containing 0-3 R₁₃, C₃-C₁₀ heterocyclyl containing 0-3R₁₃, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₅-C₁₀ cycloalkenyl, C₅-C₁₀heterocycloalkenyl, C₆-C₂₀ aryl containing 0-3 R₁₄, or C₆-C₂₀ heteroarylcontaining 0-3 R₁₄; or (ii) —C(O)R₁₀, wherein R₁₀ is defined as above;or (iii) —C(R₁₀)₂(R₁₅), wherein R₁₀ is defined as above; R₁₅ is hydrogenor R₁₀; R₂ is hydrogen; R₃, R₄, R₅, R₆, and R₇ are each independentlyhydrogen, C₁-C₆ alkyl, C₆-C₁₂ aralkyl, or C₁-C₆ acyl; R₉ is a linear orbranched C₃-C₁₀₀ alkyl; R₁₁ is C₁-C₂₀ alkyl optionally substituted withhalo, hydroxy, alkoxy, amino, alkylamino, dialkylamino, sulfate, orphosphate; R₁₂ is aryl optionally substituted with halo, haloalkyl,hydroxy, alkoxy, nitro, amino, alkylamino, dialkylamino, sulfate, orphosphate; Each R₁₃ is independently halo, haloalkyl, hydroxy, alkoxy,oxo, amino, alkylamino, dialkylamino, sulfate, or phosphate; Each R₁₄ isindependently halo, haloalkyl, hydroxy, alkoxy, nitro, amino,alkylamino, dialkylamino, sulfate, or phosphate; x is 1-100; LG is halo,—OSO₂R₁₆, B(OH)₂, or

and R₁₆ is alkyl, haloalkyl or aryl optionally substituted with alkyl,halo or nitro.
 25. A pharmaceutical composition comprising a compoundaccording to claim 1 and a pharmaceutically acceptable carrier.
 26. Anisolated antigen presenting cell for presenting a lipid antigen to NKTcells, the antigen presenting cell comprising CD1d molecules loaded witha compound according to claim 1.